Method of treating mammals with genistein and/or genistein analogues

ABSTRACT

The present invention is concerned with a method of controlling the plasma genistein concentration in mammals in order to avoid activation of the peroxisome proliferator-activated receptor γ (PPARγ), said method comprising the steps of: a. assessing the genistein blood serum concentration of the mammal; b. if needed, administering to said mammal a genistein component in an amount sufficient to maintain the genistein blood serum concentration at a level between 0.02 and 3 μM during at least 8 hours, preferably at least 16 hours of each day; c. repeating steps a. and b. during a period of at least 30 days with intervals of no more than 3 days. The present method is particularly suited for preventing obesity and diseases or conditions in which bone tissue is lost.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of in vivo administration ofdefined amounts of genistein in order to maintain the plasma genisteinconcentration within defined ranges in mammals. More particularly, thepresent invention is concerned with a method of controlling the plasmagenistein concentration in order to stimulate osteogenesis whilst at thesame time preventing adipogenesis by avoiding activation of theperoxisome proliferator-activated receptor γ (PPARγ).

The present method is particularly suited for preventing obesity anddiseases or conditions in which bone tissue is lost (such as inosteoporosis, Paget's disease, osteolytic metastasis in cancer patients,osteodystrophy in liver disease, altered bone metabolism caused by renalfailure or haemodialysis, bone fracture, bone surgery, pregnancy,lactation, anorexia nervosa, or immobile, non-weight bearing,malnourished or weight losing persons).

BACKGROUND OF THE INVENTION

Isoflavones have been advocated for the prevention of a variety ofdiseases. The actual functionality of these isoflavones is howeverpoorly understood. They are known to bind to the two known estrogenreceptors (ER's) ERα and ERβ, mediating estrogenic effects (Anderson,2001). In addition, genistein is also an inhibitor of protein tyrosinkinase. Isoflavones have however also other anti-estrogenic and non-ERdependent effects. The mechanisms of these effects are still unknown(Anderson, 2001; Aldercreutz and Mazur, 1998; Kim et al., 1998).

The optimum dosage for the prevention of bone loss and obesity has notbeen determined, since dose-effect relationships have not been wellestablished in vivo or in vitro. This is believed to be due to the factthat many known in vitro systems have a low reproducibility and/orsensitivity.

Anderson et al., Proc. Soc. Exp. Biol. Med. 1998: 217(3), 345-350,describe a positive effect of genistein on the retention of bone mass inovariectomised, lactating rats at dosages of 6.2-19.7 μmole genisteinper kg bodyweight per day. A lower response is reported for higherdosages (61.7 μmole genistein per kg bodyweight per day) in addition toa negative impact on uterine weight. The genistein dosages employed inthis study are extremely high and exceed the daily amounts of genistein,calculated on bodyweight, that are normally consumed by humans.

Nuttall et al., “Is there a therapeutic opportunity to either prevent ortreat osteopenic disorders by inhibiting marrow adipogenesis”, Bone27(2), (2000), 177-184, present a review that explores the stromalcell's differentiation plasticity in the context of osteoporosis andother metabolic bone disorders. A reciprocal relationship is postulatedto exist between the adipocyte and osteoblast phenotypes. The signaltransduction pathways implicated in this process are evaluated aspotential targets for therapeutic intervention and drug design. It isobserved in the article that in vitro and in vivo studies demonstratethat ligands binding to the PPARs, glucocorticoid, estrogen, androgenand vitamin D₃ receptors regulate bone marrow stromal cell adipogenesisand osteogenesis. In addition it is observed that drugs belonging to thethiazolidinediones promote bone marrow stromal cell adipogenesis at theexpense of osteogenesis.

The family of transcription factors known as the peroxisomeproliferator-activated receptors (PPARs) plays a central role inregulating the storage and catabolism of dietary fats. The PPARs werecloned less than a decade ago as orphan members of the nuclear receptorgene family that includes the receptors for the steroid, retinoid andthyroid hormones. It has been suggested to use PPARs as moleculartargets for the development of drugs to treat human metabolic diseases.

There are 3 PPAR subtypes, which are the products of distinct genes andare commonly designated PPARα, PPARγ and PPARδ. In common with othermembers of the nuclear receptor gene family, the PPARs areligand-activated transcription factors. The binding of agonist ligandsto the receptor results in changes in the expression level of mRNAsencoded by PPAR target genes. This process is known as“transactivation”, and cell-based assays have been developed which canbe used to monitor this functional activity.

The biology of the PPARs has been driven, in large part, by theavailability of potential and selective ligands for the receptors.Through the use of binding and functional assays, several groups havereported the identification and optimization of PPAR ligands for each ofthe 3 subtypes. These chemical tools have been used in a “reverseendocrinology” approach to uncover the role of the PPARs in humanphysiology and disease processes.

PPARγ is the most extensively studied of the 3 PPAR subtypes to date.PPARγ is a critical transcription factor in the regulation of adipocytedifferentiation. It has been found that forced expression of PPARγ infibroblasts in the presence of weak PPARγ activators resulted indifferentiation of the cells to adipocytes. Data available to date showthat PPARγ plays a pivotal role in the adipogenic signaling cascade andalso suggest that the receptor can influence the production and cellularuptake of its own activators.

Known natural PPARγ ligands include thiazolidines, a number ofpolyunsaturated fatty acids, eicosanoid derivatives and J-series ofprostaglandins derived from PGD₂, that binds and activates the receptorat micromolar concentrations.

Although the stimulation PPARγ is associated with decreased osteogenesisand increased adipogenesis, the activation of PPARγ also leads to healthbenefits. The most extensively studied therapeutic utility for PPARγagonists has been in the treatment of type 2 diabetes. In additionstudies have been conducted that show that PPARγ agonists decreaseplasma levels of triglycerides, cholesterol, and free fatty acids invarious animal models of dyslipidemia. Furthermore there are indicationsthat PPARγ agonists may have antihypertensive, anti-inflammatory andanti-tumor effects.

Liang et al, “Suppression of inducible cyclooxygenase and nitric oxidesynthase through activation of PPARγ by flavonoids in mousemacrophages”, FEBS Letters 496 (2001) 12-18, report that the flavonoidsapigenin, chrysin and kaempferol may act as allosteric effectors thatare able to bind and activate PPARγ. Genistein did in this study howevernot stimulate PPARγ activity at a concentration of 10 μM. PPARγ bindingstudies for genistein were not performed. It is suggested that theseflavonoids might have therapeutic applications in inflammatory diseases,such as atherosclerosis and rheumatoid arthritis.

U.S. Pat. No. 5,506,211 (The UAB Research Foundation) describes a methodfor use in inhibiting osteoclast activity which leads to bonedegradation, comprising contacting an osteoclast with a compositioncomprising an amount of a genistein-glucoside conjugate that occursnaturally in soy. The advocated daily doses of genistein-glucosideconjugate are in the range of 2-50 mg.

U.S. Pat. No. 5,953,996 (Taishi Food Company) describes a method foraccelerating ossification and preventing reduction of bone salt contentwhich comprises administering to a mammal in need thereof an effectiveamount of a composition containing synergistic effective amounts ofgenistein and a zinc salt. It is observed that the inventors have foundthat genistein and zinc may enhance osteogenesis. The compositionsdescribed are preferably ingested so that the concentration of genisteinin blood in one hour after ingestion may be 10⁻⁷ M or more.

WO 99/66913 (Sigma-Tau Healthscience) describes a composition, whichcomprises as active ingredients propionyl L-carnitine and genistein forthe prevention and/or therapeutic treatment of osteoporosis andmenopause syndrome.

WO 01 74345 A (Ingram Jonathan) describes a method of suppressing weightgain, inducing weight loss, or imparting a feeling of gastric fullness,comprising administering at least one isoflavone, such as genistein. Theisoflavone may be administered in a range of 5-500 mg/day.

U.S. Pat. No. 6,326,366 (Protein Technologies International) isconcerned with a hormone replacement theray regimen comprisingco-administering a therapeutically effective amount of a combination ofmammalian estrogen and isoflavone (e.g. genistein). The advocated dailydosage of isoflavone is in the range of 20-1000 mg.

EP-A 0 829 261 relates to a composition for fat-degradation in a fatcell which comprises at least one isoflavone and/or a derivativethereof. The isoflavone is suitably selected from the group consistingof daidzein, daidzin, genistein and genistin. In the patent application,a dosage dependent stimulation of lipolysis in adipocytes is observedafter exposure to 3 to 100 micro M genistein. No effects were observedat 0.3 and 1 μM.

Harmon et al. (Am J Physiol Cell Physiol. 2001 April;280(4):C807-13)report the results of a study into the differential effects offlavonoids on 3T3-L1 adipogenesis and lipolysis. It is concluded thatthe flavonoids genistein and naringenin inhibit proliferation ofpreadipocytes in a time- and dose-dependent manner. When added to 2-daypostconfluent preadipocytes at the induction of differentiation,genistein was found to inhibit PPAR-γ expression. On page C812 it isobserved “Given that genistein promotes lipolysis and inhibitsadipogenesis in cell culture, we anticipate that genistein will actsimilarly in vivo and potentially promote loss of body fat”. It isfurthermore stated: “a non-toxic pharmacological dose of genistein, 8mg/kg body weight, elevates serum genistein to the 10 μM range . . . , alevel sufficient to inhibit adipogenesis in 3T3-L1 cells.

Dang et al. (J. of Bone and Mineral Res., 2000 September;15(1), S496)describe the results of a study into the effects of genistein and17β-estradiol on osteoblastogenesis and adipogenesis. Two of the presentinventors are mentioned as authors of this publication. The abstractpresents data on the effects of exposure of KS485 cells to lowconcentrations of genistein. Although this experimental work provided afoundation for the present invention, it was not known at the time thathigher concentrations of genistein produce an adverse and oppositeeffect to those observed at lower concentrations. Wang et al. (J. Biol.Chem. 1999 Nov. 5;274(45):32159-66) report the results of a study thatshow the ability of a tyrosine kinase inhibitor to block adipogenesis inresponse to dexamethasone and methylisobutylxanthine and providecompelling evidence for a central role of the tyrosine kinase Syk in theG_(S)α-mediated regulation of adipogenesis. In the article the followingobservations are made: “The dose dependence with respect to the effectsof genistein on D/M induced adipogenesis was explored. At 100 μMgenistein effectively abolishes adipogenic conversion. Both 10 and 33 μMconcentrations of the tyrosine kinase inhibitor provoke a markedinhibition of adipogenesis. at 3.3 μM, the lowest concentration tested,genistein displays only a weak inhibitory influence on thedifferentiation of the 3T3-L1 cels in response to the inducers D/M. Noreferences can be found in any of the aforementioned prior art documentsas to the ability of isoflavones, and particularly of genistein, to bindand activate PPARγ, i.e. to act as a PPARγ agonist. Although the use ofgenistein components has been advocated for the treatment ofosteoporosis and a wide variety of other disorders and diseases, theactual functionality of these isoflavones is poorly understood. Thedose-effect relationships for genistein and osteoporosis are not wellestablished and, as will be shown below, are intrinsically flawedbecause they have not taken account of the PPARγ mediated effect ofgenistein on adipogenisis.

SUMMARY OF THE INVENTION

The inventors have discovered that genistein components may suitably beused to treat or prevent a variety of diseases including osteoporosis,obesitas and/or syndrome X (insulin resistance syndrome), provided theseisoflavones are administered in accordance with a regime that does notlead to plasma genistein levels that are sufficiently high to activatethe PPARγ receptor. Despite the large number of prior art publicationsthat deal with the favourable effect of genistein on e.g. osteogenesisand/or adipogenesis, it has not been recognized before that it iscrucial to avoid that plasma genistein levels reach a level that issufficient to activate the PPARγ receptor in order to prevent inhibitionof osteoblast activity and differentiation and to prevent stimulation ofadipogenesis. As a matter of fact some of these publications recommendgenistein dosages and plasma levels that according to the inventors'findings will produce significant adverse results. The inventors havedeveloped a highly reproducible and sensitive in vitro model that hasallowed them to discover that the effect of genistein components on e.g.osteogenesis and adipogenesis is biphasic, i.e. at plasma genisteinlevels above about 0.02 μM estrogen receptors (ER's) may be activated,leading to stimulation of osteogenesis, whilst at plasma genisteinlevels above about 4 μM the PPARγ receptor is activated, which triggersadipogenesis. By maintaining the plasma genistein levels at a levelwhere the estrogen receptors are continuously activated, whilst at thesame time avoiding activation of the PPARγ receptor, disorders such asosteoporosis and obesity may effectively be treated. The inventors havefound that at low plasma concentrations (0.02-3 μM) osteogenesis isstimulated and adipogenesis is inhibited by:

-   1. a stimulation of proliferation of osteoblastic precursor cells-   2. a stimulation of differentiation of osteoblasts at the expense of    a differentiation in the direction of adipocytes, i.e., a    stimulation of osteoblastogenesis and a inhibition of adipogenesis-   3. a stimulation of osteoblastic activity.    These effects are mediated through the ER's.

The investigators have further found that at high genisteinconcentrations (i.e. above 4 μM, especially above 6 μM), genistein bindsto PPARγ, i.e. acts as a true PPARγ agonist. This results in theinhibition of cell proliferation and a decrease in osteoblastogenesisand osteogenesis. At the same time the adipogenesis increases. Inaddition to its activation of PPARγ activity through direct binding,genistein inhibits MAPK activity, thereby potentiating PPARγ activity.Although at high concentrations ER's are stimulated by genistein, PPARγactivation dominates the biological outcome, i.e. an increase ofadipogenesis at the expense of osteoblastogenesis. This results in an“overruling” of the ER mediated effects by PPARγ mediated effects. Inaddition, there is cross talk between the activity of the receptors,mutually inhibiting each other activities.

It is noted that the aforementioned study by Anderson et al. suggeststhat high dosages of genistein are less effective than low andintermediate dosages. However, both the intermediate and high dosagesemployed in said study exceed the dosages encompassed by the presentinvention.

The aforementioned biphasic dose response of genistein has obviousimplications for the use of genistein in the prevention of diseases thatare not only estrogen dependent but also dependent on PPARγ activation.In particular in cases where the higher doses lead to undesiredinhibition of cellular proliferation and differentiation (i.e, towardsadipocytes), care must be taken to ensure that genistein serum levelsare maintained within the bandwidth that is defined by the lowest dosethat is effective in stimulating an ER, and the lowest dose that iseffective in stimulating PPARγ activity. If the doses employed areinsufficient to activate an ER (i.e., below 0.02 μM in plasma) therewill be no physiological effect. If, however, these doses are too high(i.e., above 4 μM), PPARγ will be activated and the actual effectobserved may well be the exact opposite of the effect intended.

Since genistein occurs in varying amounts in a wide array of foodproducts, it is important to realize that blood serum genistein levelsare greatly affected by an individual's diet and/or use of nutritionalsupplements. It has been reported that the plasma concentration ofgenistein is relatively low and generally less than 20 nM in humansconsuming diets without soy. In contrast, it can reach almost 5 μM inthe plasma of Japanese who consume high amounts of soy products. Thus,in a method that aims to maintain the blood serum genistein level at alevel where it is capable of stimulating osteogenesis and inhibitingadipogenesis, it is crucial to adapt the amount in which genistein isadministered to the diet of the individual, to avoid exceeding the upperlevel above which the opposite effect is observed, due to the activationof PPARγ.

DETAILED DESCRIPTION OF THE INVENTION

Consequently, the present invention is concerned with a method ofcontrolling the plasma genistein concentration in mammals in order toavoid activation of the peroxisome proliferator-activated receptor γ(PRARγ), said method comprising the steps of:

-   a. assessing the genistein blood serum concentration of the mammal;-   b. if needed, administering to said mammal a genistein component in    an amount sufficient to maintain the genistein blood serum    concentration at a level between 0.02 and 3 μM, preferably between    0.05 and 2 μM, during at least 8 hours, preferably at least 16 hours    of each day;-   c. repeating steps a. and b. during a period of at least 30 days    with intervals of no more than 3 days.

The term “genistein blood serum concentration” refers to the combinedblood serum concentration of pure genistein and certain metabolites ofgenistein. A suitable method for determining the (combined) genisteinblood serum concentration is described by Setchell et al, 2001 J. Nutr.131:1362-1375S. This method enables the determination of pure genisteinas well as of genistein metabolites such as genistein glycons.Throughout this document the terms genistein blood serum concentrationand plasma genistein concentration are used interchangeably.

The term “genistein component” encompasses genistein as well asprecursors of genistein that are capable of liberating genistein whenused in accordance with the present method and derivatives of genisteinthat display a similar functionality, i.e. that are also capable ofactivating an ER and PPARγ at similar blood serum concentrations.Whenever reference is made in this document to genistein blood serumconcentration it should be understood that this not only relates to theblood serum concentration of genistein per se, but also to the combinedblood serum concentration of genistein and of genistein derivatives thatdisplay similar in vivo functionality.

The in vivo half-life time of genistein is between 5 and 10 hours and,amongst others, dependent on sex and individual physiology. This is whyit is preferred to administer genistein component at least once a day(if needed). If longer administration intervals are employed, relativelyhigh doses are necessary to constantly maintain the genistein bloodserum concentration at a sufficiently high level. Such relatively highdoses increase the risk that genistein blood serum concentration willexceed 3 μM and thus the risk that the administration of the genisteincomponent will have the opposite effect of what is intended. It isparticularly preferred to administer the genistein component at leasttwice daily.

In a special embodiment of the present invention the method comprisesthe once daily administration of a slow release formulation comprisingthe genistein component. The use of a slow release formulation offersthe advantage that the genestein blood serum concentration may bemaintained at a relatively constant level without the need ofadministering the genistein component 2, 3 or even more times per day.

It should be understood that if the assessment of the genistein bloodserum concentration in step (a) shows said concentration to exceed 3 μM,no genistein will be administered until the day when the estimatedconcentration is within the range of 0.02 and 3 μM. Thus, the presentmethod also encompasses an administration regimen comprising intervalsduring which no genistein is administered.

The assessment of the genistein blood serum concentration of a mammalmay be done by means of analysis or alternatively by estimating saidconcentration on the basis of the recent diet of said mammal. Because itis often impractical to carry out regular blood serum analyses to assessthe blood serum genistein concentration, it is preferred to assess thegenistein blood serum concentration on the basis of the mammal's recentdiet. Here by the recent diet is meant the assortment of foodstuffs andnutritional supplements that have been consumed during a period of nomore than 4 days prior to the assessment. Preferably the aforementionedassessment is done on an at least once daily basis.

In order to maintain genistein blood serum concentration below 3 μM, thegenistein component should generally be administered in an amount whichis equivalent to a total daily oral dosage of between 0 and 6 μmole perkg bodyweight, preferably equivalent to a daily oral dosage of between 0and 2.5 μmole per kg of bodyweight. In most individuals the minimumamount of genistein component that needs to be administered to maintaina sufficiently high genistein blood serum concentration exceeds theequivalent of a daily oral dosage of 0.04 μmole per kg of bodyweight.

In accordance with the present invention the genistein component may beadministered in a variety of ways, e.g. enterally or parentally. Mostpreferably the genistein component is administered orally. The genisteincomponent may suitably be administered orally in the form of a sustainedrelease formulation, which releases the genistein component at such arate that the genistein blood serum concentration remains within theaforementioned critical ranges. This may be achieved by selecting asustained release formulation that releases less than 1.3 μmole, morepreferably less than 1 mmole genistein component per kg of bodyweightduring the first 3 hours after administration.

Preferably the genistein component is administered in the form of anutritional or pharmaceutical dosage unit that comprises at least 2.8μmole genistein component, more preferably approximately 10-400 μmolegenistein component and most preferably 50-175 μmole genisteincomponent.

Examples of pharmaceutical dosage units that may suitably be used in thepresent method include tablets, capsules, powders and the like. In anadvantageous embodiment of the invention, the pharmaceutical dosage unitis a sustained release formulation. The use of a sustained releaseformulation offers the advantage that it helps to prevent majorfluctuations in genistein blood serum concentrations. Thus the risk thatgenistein serum concentrations will move outside the desired bandwith issignificantly reduced. Examples of suitable sustained releaseformulations include tablets that comprise a core which contains thegenistein component, which core is enveloped by a semi-porous coating(e.g. an acrylate). It is also possible to include the genistein inmicrocapsules which disintegrate under predefined conditions in thegastrointestinal tract. Furthermore it is feasible to incorporate thegenistein component in a pellet which, during passage through thegastointestinal tract disintegrates slowly because of a combination ofits size and water solubility. Examples of suitable nutritional dosageunits that may suitably be used in accordance with the present inventioninclude cereals, nutritional bars and nutritional drinks.

In accordance with the present method, the genistein component issuitably administered within one hour after the last assessment of thegenistein blood serum level. More preferably said administration occurswithin half an hour after the assessment.

In a preferred embodiment of the invention step (b) comprisesadministering the genistein component, if needed, in an amountsufficient to maintain the genistein blood serum concentration at alevel between 0.02 and 3 μM. Preferably the genistein component isadministered in an amount sufficient to initially raise the genisteinblood serum concentration to at least 0.5 μM, preferably at least 0.7μM, and maintaining said concentration at a level of at least 0.3 μM,more preferably at least 0.4 μM until the next administration.

The present method may suitably be employed to prevent diseases such asosteoporosis and/or obesity by the stimulation of osteogenesis and theinhibition of adipogenesis. The present method is particularly effectivein the prevention and treatment of osteoporosis, includingpostmenopausal osteoporosis, as maintenance of the genistein blood serumconcentrations within the advocated bandwidth of 0.02-3 μM, will notonly stimulate osteogenesis, but also inhibit adipogenesis. Theinhibition of adipogenesis may effectively be used to suppress theformation of body fat in the context of the (prophylactic) treatment ofobesity.

The effects of the present invention are most pronounced in tissue inwhich both estrogen and PPARγ receptors are abundantly present. Examplesof such tissues are breast, bone marrow, prostate, vagina, ovaries,uterus and adipose tissue, e.g. in the brain, liver and pancreas.

In order to ensure that the beneficial effects of the present methodwill last, it is advocated to continue the present method for a periodof at least 6 months, more preferably at least 12 months.

A reliable indication as to whether the amount of genistein componentthat has been administered is sufficiently high can be obtained fromassessing the impact on osteogenesis. Preferably, the genisteincomponent, if needed, is administered in an amount sufficient to raisethe genistein blood serum concentration to a level where osteogenesis isstimulated. Most preferably osteogenesis is increased by at least 5%relative to the level observed prior to the administration of thegenistein component.

It is an essential aspect of the present method that the daily dose ofgenistein component is adjusted to the intake of genistein through thedaily diet. In particular in those cases where the diet leads tosubstantial fluctuations in the blood serum genistein concentration, thepresent method can produce particularly good results. Preferably, duringthe period of at least 30 days, the amount of genistein component thatis administered on a daily basis fluctuates between a minimum and amaximum amount that differ by at least a factor 3. More preferably sucha differential factor is observed without there being a day during said30 days when no genistein component was administered.

Even though the present method encompasses regimens with administrationfree intervals, in a preferred embodiment the genistein component isadministered at least once a week. More preferably the genisteincomponent is administered at least 3 days a week. Most preferably thegenistein component is administered at least once daily during theperiod of at least 30 days. Particularly preferred is a method whereinthe genistein component is administered at least twice daily. Apreferred mode of twice daily administration comprises theadministration at a time interval of between 8 and 16 hours. The dosageamounts are preferably adjusted to the time interval between theadministrations, meaning that if the next administration is foreseenafter e.g. 16 hours, a dose is used which is twice as high as the dosethat would be used in case the next administration is foreseen in 8hours. In a particularly preferred administration regime one dose isadministered in the morning (e.g. between 6:00 and 10:00 a.m.) andanother dose in the evening (e.g. between 5:00 and 9:00 p.m.). Suitablyat least 60 mol % of the daily dosage of the genistein component isadministered in the evening.

In a preferred embodiment of the invention the genistein componentcomprises one or more substances represented by the following formula:

wherein R₅, R₇, and R_(4′) are independently a hydrogen atom; asaturated or unsaturated, linear, branched or cyclic, optionallysubstituted, alkyl group having from 1 to 6 carbon atoms; an acyl groupwith a saturated or unsaturated, linear, branched or cyclic, optionallysubstituted, alkyl radical having from 1 to 8 carbon atoms; a sulphategroup; or a mono-, di- or trisaccharide group.

Preferably R₅, R₇, and R_(4′) are independently a hydrogen atom; asaturated, linear or branched alkyl group having from 1 to 3 carbonatoms; an acyl group with a saturated alkyl radical having from 1 to 3carbon atoms; a sulphate group; or a mono-, di- or trisaccharide group.More preferably R₅, R₇, and R_(4′) are independently a hydrogen atom; amethyl group; an acyl group with a saturated alkyl radical having from 1to 3 carbon atoms; or a mono- or disaccharide group.

Genistein components wherein at least one of R₅, R₇, and R_(4′) is analkyl or acyl group, offer the advantage of improved stability andhigher activity. Genistein components wherein at least one of R₅, R₇,and R_(4′) is a mono-, di- or trisaccharide group (i.e. glycosylatedgenistein components) first need to be metabolised into aphysiologically active metabolite. Hence such derivatives can offer theadvantage of delayed impact.

Glycosylated genistein components that are particularly suited for usein the present method include 7-O-βD-glycoside (genistin). In case thepresent method employs glycosylated genistein component, it is preferredthat at least 50 wt. % of the glycosylated genistein component is a7-O-βD-glycoside.

Such a delayed impact is particularly advantageous if the genisteincomponent is administered in the evening, particularly between 5:00 and9:00 p.m. In the morning, however, i.e. between 6:00 and 10:00 a.m., itis advantageous to administer a largely non-glycosylated genisteincomponent as this will result in a more predictable response in terms ofblood serum genistein levels. Hence, in a preferred embodiment thepresent method comprises the steps of administering:

-   a) at least 0.02 μmole genistein component per kg of bodyweight    between 5:00 and 9:00 p.m, wherein at least 70 mol. % of the    genistein component is glycosylated; and-   b) at least 0.02 μmole genistein component per kg of bodyweight    between 6:00 and 10:00 a.m., wherein at most 30 mol. % of the    genistein component is glycosylated.

Genistein components may suitably be obtained from natural sources suchas soy flour or fermented soy. The application of synthetic genisteinand particularly synthetic derivatives of natural genistein is alsoencompassed by the present invention. Most preferably the presentinvention employs a genistein component that is obtained from a plantmaterial, preferably the genistein component is obtained from a plantextract, more particularly a proteinaceous plant extract. As mentionedherein before blood serum genistein concentrations are strongly affectedby an individual's diet. In particular soy based food products can havea serious impact on said serum concentrations. Consequently, in apreferred embodiment of the present method, step (a) comprisesestimating the blood serum genistein concentration on the basis of theamounts of soy based staple food products that were consumed by themammal during the previous 24 hours. By providing information about thegenistein content of such staple food products and taking into accountthe quantities that were consumed within a given period, it is possibleto estimate the genistein blood serum concentration and consequently theamount of genistein component that needs to be administered to maintainoptimum genistein blood serum level.

In the present method it may be advantageous to co-administer knownactive principles against osteoporosis and obesity. Suitable activeprinciples against osteoporosis that may advantageously be administeredin combination with the genistein component include polyphenols, vitaminK, zinc, calcium, and/or vitamin D. Preferably the amount of vitamin Dwhich is co-administered does not exceed 1 mg per day, more preferablyvitamin D is co-administered in an amount of between 2-10 μg per day.The daily amount of calcium is advantageously kept between 60-1000 mg,preferably between 80-800 calcium. Zinc is suitably administered in apharmaceutically acceptable form in an amount of no more than 40 mg perday, preferably no more than 30 mg per day. Per dosage (i.e.administration event) the amount of zinc is preferably kept below 15 mgbecause of the adverse taste effect observed with higher doses.Preferably zinc dosages exceed 5 mg per day. In the prevention ofobesity, genistein may be combined with hydroxy citric acid andgrapeseed extract.

Another aspect of the invention relates to a method of treating orpreventing osteoporosis and/or obesity in mammals, said methodcomprising the steps of administering:

-   a) at least 0.02 μmole genistein component per kg of bodyweight    between 5:00 and 9:00 p.m, wherein at least 70 mol. % of the    genistein component is glycosylated; and-   b) at least 0.02 μmole genistein component per kg of bodyweight    between 6:00 and 10:00 a.m., wherein at most 30 mol. % of the    genistein component is glycosylated.

The advantages of such an administration regimen have been explainedherein before. The preferred embodiments of this method are essentiallyidentical to those described in relation to the aforementioned method ofcontrolling the plasma genistein concentration in order to stimulateosteogenesis and to prevent adipogenesis through the activation of PPARγin mammals.

Another aspect of the invention relates to a pharmaceutical ornutritional kit comprising one or more discrete oral dosage unitscontaining 1.4-200 μmole genistein component, wherein at least 70 mol. %of the genistein component is glycosylated; and one or more discreteoral dosage units containing 1.4-200 μmole genistein component, whereinat most 30 mol. % of the genistein component is glycosylated. Preferablythe oral dosage units contain at least 2.8 μmole of the genisteincomponent, more preferably at least 10 μmole. The amount of genisteincomponent in the dosage unit preferably does not exceed 150 μmole. Thepresent kit is preferably provided with instructions for the userindicating that the oral dosage units should be administered inaccordance with the method as described herein before. In the presentkit, the number of oral dosage units containing at least 70 mol. % ofglycosylated genistein and the number of oral dosage units containingnot more than 30 mol. % of glycosylated genistein are preferablyvirtually the same, more preferably, they are exactly the same.

Yet another aspect of the invention relates to an oral dosage unitcontaining at 1.4-200 μmole genistein component, 2-10 μg vitamin D and25-3000 μg, preferably 80-800 μg vitamin K. Vitamin D is advantageouslyemployed in the treatment or prevention of osteoporosis as this vitaminis known to enhance calcium absorption. However, vitamin D is alsobelieved to stimulate the formation of osteoclasts. As explained before,the present method aims to promote the formation of osteoblasts whilstat the same time inhibiting the formation of osteoclasts. Thus, theadvantageous effect of vitamin D on calcium absorption is offset by itsundesirable effect on osteoclast formation. This undesirable effect,however, may be counteracted effectively by incorporating a sufficientamount of vitamin K. Additional advantages associated with the use ofvitamin K reside in the vitamins ability to inhibit stimulation of PPARγand its stimulating effect on osteoblast formation.

Preferably the oral dosage units contain at least 2.8 μmole of thegenistein component, more preferably at least 10 μmole. The amount ofgenistein component in the dosage unit preferably does not exceed 150μmole. In a preferred embodiment, the oral dosage unit is a discretesolid or semi-solid oral dosage unit such as a tablet or a bar. Theamount of genistein component in the oral dosage is preferably withinthe range of 10-400 μmole.

The invention is further illustrated by means of the following examples.

EXAMPLES Example 1

Effect of Genistein on Osteogenesis

KS 483 cells were continuously treated for 21 days with variousconcentrations of genistein from day one onwards. Genistein had clearbiphasic effects on osteogenesis. The stimulatory effects of genisteinon alkaline phosphatase (ALP) activity, nodule formation and Cadeposition were found in the range of 0.1 μM to 10 μM, with a maximaleffect at 1 μM. In contrast, the inhibitory effects of genistein on ALPactivity, nodule formation and Ca deposition occurred at concentrationsof 25 μM or higher. An increase of bone formation at 1 μM and a decreaseof bone formation at 25 μM were further confirmed by mRNA expression ofthe osteoblastic markers, Cbfal, osteocalcin and parathyroid hormone(PTH)/parathyroid hormone related peptide (PTHrP) receptor.

Similar stimulatory and inhibitory effects of genistein on boneformation were also observed in mouse and human bone marrow cellcultures. Like in KS483 cells, the stimulatory effects on ALP activityand Ca deposition in mouse bone marrow cultures occurred atconcentrations between 0.1 μM and 10 μM, whereas the inhibitory effectswere also found at the concentrations of 25 μM or higher. In human bonemarrow cell cultures, the stimulatory effects was found at 0.001 μM,whereas the inhibitory effects were observed above 4 μM.

These findings demonstrate that genistein affects osteogenesis ofprogenitor cells in a biphasic way, i.e., an increase of osteogenesis atlow concentrations and an inhibition of osteogenesis at highconcentrations.

Effect of Genistein on_adipogenesis

In contrast to osteogenesis, genistein concurrently inhibited orstimulated adipogenesis in KS483 cells, mouse and human bone marrowcells. Dose-related adipogenic responses in KS483 cells treated withgenistein from day 1 onwards showed that genistein decreased adipocytenumbers in the range of 0.1 to 1 μM, whereas it increased adipocytenumbers at concentrations of 10 μM or higher.

These inhibitory and stimulatory effects of genistein on adipogenesis inKS483 cells were further confirmed by mRNA expression of the adipocytemarkers, PPARγ2, adipocyte protein 2 (aP2) and lipoprotein lipase (LPL).Adipogenic responses of mouse bone marrow cells to different doses ofgenistein were obtained. Mouse bone marrow cultures exposed to genisteinconcentration of 25 μM or higher were not confluent and there were noadipocytes during the cultures. However, an increase in adipocytenumbers was observed at concentrations above 4 μM, whereas a decrease inadipocyte numbers was found at the concentrations of 0.1 and 1 μM.

Similarly, in human bone marrow cultures exposed to different doses ofgenistein, an increase in adipocyte numbers was observed at theconcentration of 10 μM, whereas a decrease of adipocyte numbers wasobserved at the concentrations of 0.001, 0.01 and 0.1 μM.

Clearly, these data show that genistein affects adipogenesis ofprogenitor cells in a biphasic way, i.e. an inhibition of adipogenesisat low concentrations and a stimulation of adipogenesis at highconcentrations.

Mechanism of Genistein Action on Adipogenesis and Osteogenesis: Effectsof Genistein on Transactivation of PPARγ, Binding and MAPK Activity.

KS483 cell were transiently transfected with a luciferase reporterconstruct containing two copies of a consensus estrogenic receptorresponsive element (ERE) inserted in front of TATA or five copies of aconsensus PPARy responsive element (PPRE) inserted in front of TATA orempty TATA-luciferase reporter along with expression plasmids encodinghuman PPARγ2. A dose-dependent increase of ERE reporter activity wasfound to occur in the genistein concentration range of 0.1 μM to 50 μM.Similarly, a dose-related increase of PPRE reporter activity was foundin the range of 1 μM to 50 μM.

The dose-related increase of PPRE reporter activity by genistein wasalso observed when a luciferase reporter construct containing threecopies of a consensus PPRE inserted in front of a minimal thymidinekinase promoter. To determine whether genistein activates PPARγ throughdirect interaction with this receptor, we performed membrane-bound PPARγbinding assay. Genistein was found to have a measurable Ki of 5.7 μM,which is comparable to the known PPARγ ligand LY 171883 (4.2 μM) andother ligands reported to bind at the micromolar range.

These data demonstrate that genistein can interact directly with thePPARγ ligand binding domain and can thus be defined as a PPARγ ligand.It has been shown that phosphorylation of PPARγ by p42 and p44 mitogenactivated protein kinase (MAPK) regulates PPARγ activity. We thereforeperformed western blot to test whether genistein at the micromolarconcentrations inhibit p42 and p44 MAPK activity. An inhibition of p42and p44 MAPK activity was found to occur in the micromolar range. Ourresults demonstrate that the transcriptional activation of ERE and PPREby genistein is dose-dependent but not biphasic. An inhibition of p42and p44 vMAPK activity is likely to potentiate PPARγ activity.

In addition, we showed here that there is interaction between ER's andPPARγ, possibly responsible for the antiestrogenic effects of genistein.We showed that a co-transfection of PPARγ2 decreased ERE reporteractivity when KS483 cells were exposed to the same concentrations ofgenistein. These results suggest that a competition of the common ligandgenistein might occur between ER's and PPARγ. It is also possible thatan activation of PPARγ down regulated transcriptional activity ofestrogen, i.e. there is cross-talk between ER's and PPARγ.Co-transfection of ERα in KS483 cells exposed to genistein resulted in adown regulation of PPRE reporter activity. These results indicate thateither a competition for the same ligand occurred between ER's and PPARγor a suppressive function of ERα on PPARγ transcriptional activity.Different from the biphasic effects of genistein on osteogenesis andadipogenesis of KS483 cells, both ERE and PPRE reporter activitiesshowed a dose-related increase and reach the maximal level at genisteinconcentration of 50 μM. The difference between biological effects andthe transcriptional activation indicates that biological effects ofgenistein depend on the balance of an activation between ER-dependentpathway and PPARγ-dependent pathway. Ligand concentration thus plays acrucial role in determining the degree of activation of ER's and PPARγ.

In conclusion, our results demonstrate that the transcriptionalactivation of ERE and PPRE by genistein is dose-dependent but notbiphasic and that there are cross talks between ER's and PPARγ.

Example 2

Two different types of tablets are prepared for use in a method oftreating osteoporosis and/or obesity. One tablet is designed foradministration in the morning between 6:00 and 10:00 a.m. (“morningtablet”), the other is other tablet is intended for administration inthe evening between 5:00 and 9:00 p.m. (“evening tablet”).

The formulations of these 2 tablets are as follows:

Morning Tablet (1 gram): Genistein   14 mg Vitamin K  0.1 mg Vitamin D  5 μg Calcium carbonate  500 mg Excipient remainder

Evening Tablet (2 gram): Soy extract  500 mg (36 mg, mainlyglycosylated, genistein) Vitamin K  0.1 mg Vitamin D   5 μg Calciumcarbonate  400 mg Zinc sulphate monohydrate   66 mg Excipient remainder

Example 3

A sustained release tablet is prepared for use in a method of treatingosteoporosis and/or obesity from the following ingredients: Tablet coreGenistein   30 mg Metolose 60SH4000 189.1 mg Magnesium stearate  0.9 mgSubtotal 220.0 mg Coating ETHOCEL 10 (Dow Chemcial)  5.0 mg Polyethyleneglycol 6000  0.5 mg Subtotal  5.5 mg Total 225.5 mgTo the genistein are added Metolose 60SH4000 and magnesium stearate, andthe mixture is thoroughly mixed. The mixture is directly compress-formedby a continuous compressor equipped with a 8 mm diameter, 6.5 R pounderunder the main pressure of 1 ton into tablets each weighing 220 mg.Then, the obtained base tablets are placed in a pan coater (FreundIndustry) and a solution of ETHOCEL 10 and polyethylene glycol 6000 in amixture of water-ethanol (1:9) is sprayed thereon, followed by drying togive the filmn coated tablets.

1-15. (canceled)
 16. A method for the treatment or prevention ofosteoporosis and/or obesity, while preventing a decrease in osteogenesisand an increase in adipogenesis, said method comprising the steps of: a)assessing the genistein blood serum concentration of a mammal; b) if theblood serum level of a) is below 3 μM, administering to said mammal agenistein component in an amount sufficient to maintain the genisteinblood serum concentration at a level between 0.02 and 3 μM during atleast 8 hours, preferably at least 16 hours of each day; if the bloodserum level of a) exceeds 3 μM, not administering to said mammal agenistein component until the blood serum level is below 3 μM; c)repeating steps a. and b. during a period of at least 30 days withintervals of no more than 3 days.
 17. A method according to claim 16,whereby the genistein blood serum concentration in step a. is assessedon the basis of the recent diet of said mammal.
 18. A method accordingto claim 16, whereby the genistein component in step b. is administeredto the mammal in an amount sufficient to raise the genistein blood serumconcentration to a level where osteogenesis is stimulated.
 19. A methodaccording to claim 16, whereby the genistein component is administeredin step b. in an amount which is below the amount that would increasethe genistein blood serum concentration to a level where activation ofPPARγ causes adipogenesis.
 20. A method according to claim 16, wherebyduring the period of at least 30 days the amount of genistein componentthat is administered on a daily basis fluctuates between a minimum and amaximum amount that differ by at least a factor
 3. 21. A methodaccording to claim 16, whereby the genistein component is administeredat least once a week.
 22. A method according to claim 16, whereby thegenistein component comprises one or more substances represented by thefollowing formula:

wherein R₅, R₇, and R_(4′) are independently a hydrogen atom; asaturated or unsaturated, linear, branched or cyclic, optionallysubstituted, alkyl group having from 1 to 6 carbon atoms; an acyl groupwith a saturated or unsaturated, linear, branched or cyclic, optionallysubstituted, alkyl radical having from 1 to 8 carbon atoms; a sulphategroup; or a mono-, di-or trisaccharide group.
 23. A method according toclaim 16, whereby the genistein component is administered orally.
 24. Amethod according to claim 16, whereby step a. comprises estimating theblood serum genistein concentration on the basis of the amounts of soybased staple food products that were consumed by the individual duringthe previous 24 hours.
 25. A method according to claim 16, whereby thegenistein component is administered in an amount equivalent to a dailyoral dosage which is within the range of 0 to 3 μmoles per kgbodyweight.
 26. A method of for treating or preventing osteoporosisand/or obesity in mammals, said method comprising the steps ofadministering: a) at least 0.02 mmole genistein component per kg ofbodyweight between 5:00 and 9:00 p.m, whereby at least 70 mol. % of thegenistein component is glycosylated; and b) at least 0.02 μmolegenistein component per kg of bodyweight between 6:00 and 10:00 a.m.,whereby at most 30 mol. % of the genistein component is glycosylated.27. A method according to claim 26, whereby at least 60 mol % of thedaily dosage of the genistein component is administered in the evening.28. A pharmaceutical or nutritional kit comprising one or more discreteoral dosage units containing 1.4-200 μmole genistein component, wherebyat least 70 mol. % of the genistein component is glycosylated; and oneor more discrete oral dosage units containing at 1.4-200 mmole genisteincomponent, whereby at most 30 mol. % of the genistein component isglycosylated.
 29. An oral dosage unit containing 1.4-200 μmole genisteincomponent, 2-10 μg vitamin D and 25-3000 μg vitamin K.
 30. A methodaccording to claim 16, whereby the genistein component is in the form ofa sustained release formulation, which releases less than 1.3 μmolegenistein component per kg body weight during the first three hoursafter administration.
 31. An oral dosage unit comprising a genisteincomponent in the form of a sustained release formulation, which releasesless than 1.3 μmole genistein component per kg body weight during thefirst three hours after administration and which comprises the genisteincomponent in an amount sufficient to maintain the genistein blood serumconcentration at a level between 0.02 and 3 μM during at least 8 hours,preferably at least 16 hours of each day.
 32. The oral dosage unitaccording to claim 31, whereby said dosage unit further comprises 80-800mg calcium and 5-15 mg zinc.